Purification or Removal of Granulocyte-Colony Stimulating Factor


Granulocyte-colony stimulating factor (G-CSF) is a hormone that stimulates production of white blood cells in bone marrow. GSCFSelect is specifically designed for purification of recombinant G-CSF and is based on a highly rigid agarose base matrix that allows for high flow rates at large production scales. For a highly selective purification step, the affinity ligand is based on a single-chain antibody fragment directed against G-CSF. To facilitate binding of the target molecule, the ligand is attached to the base matrix through a hydrophilic spacer arm (Fig 3.16). The ligand is produced in a yeast expression system, where fermentation, subsequent purification, and formulation are performed in the absence of animal-derived components. The rigid agarose base matrix of GCSFSelect allows for processing of large sample volumes.

Structure of GCSFSelectFig 3.16. Structure of GCSFSelect.

Chromatography medium characteristics

Characteristics of GCSFSelect chromatography medium are shown in Table 3.18.

Table 3.18. Characteristics of GCSFSelect chromatography medium

Product Ligand Composition pH stability1 Average particle size (µm)
GCSFSelect Single-chain antibody fragment (Mr 14 400) directed against G-CSF and produced in Saccharomyces cerevisiae Ligand coupled to Capto via stable amide bonds Short term: 2 to 12
Long term: 3 to 10

1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

Purification options

GCSFSelect is available in chromatography media packs for packing in columns and in prepacked HiTrap® columns, see Table 3.19.

Table 3.19. Purification options for GCSFSelect chromatography medium and prepacked columns

  Binding capacity Maximum
operating flow
GCSFSelect G-CSF, 3.9 mg/ml medium 600 cm/h1 Media suspension ready for column packing
HiTrap® GCSFSelect, 1 ml G-CSF, 3.9 mg/column 4 ml/min Prepacked 1 ml column
HiTrap® GCSFSelect, 5 ml G-CSF, 19.5 mg/column 20 ml/min Prepacked 5 ml column

1 Flow velocity measured in a 1 m diameter column, 20 cm bed height at 20°C; buffers used had same viscosity as water at < 0.3 MPa (3 bar, 43.5 psi).

Purification examples

Figure 3.17A shows an example of a purification of G-CSF using GCSFSelect. The sample was E. coli lysate spiked with recombinant G-CSF and elution was performed using bis-Tris buffer containing 0.08% Tween and 1 M MgCl2 (pH 7.0). Fractions from the purification were further analyzed by SDS-PAGE (Fig 3.17B). The single eluted peak with high purity demonstrates the high selectivity for G-CSF of the GCSFSelect medium.

Purification of G-CSF from a G-CSF spiked E. coli lysateFig 3.17. (A) Purification of G-CSF from a G-CSF spiked E. coli lysate. (B) SDS-PAGE of the different fractions collected during purification of G-CSF using GCSFSelect.

Performing a separation

Binding buffer: PBS (10 mM sodium phosphate, 140 mM NaCl), pH 7.4
Elution buffer: 20 mM bis-Tris, 0.08% Tween 20, 1 M MgCl2, pH 7.0
  1. Equilibrate with 10 CV of binding buffer.
  2. Load the sample.
  3. Wash with binding buffer until no material appears in the eluent (monitored by UV absorption at A280 nm).
  4. Elute with 5 to 10 CV of elution buffer.


Solutions such as PAB (120 mM phosphoric acid, 167 mM acetic acid, 2.2% benzyl alcohol) in cleaning protocols are suggested. Cleaning and sanitization protocols should be designed for each process as the efficiency of the protocol is strongly associated with the sample and other related operating conditions.


Store at 4°C to 8°C in 20% ethanol.